Abstract
BackgroundA novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system.ResultsA 6,665-bp DNA region carrying just five ORFs (dndA-E) was defined as the sole determinant for modification of the DNA backbone in S. lividans to form phosphorothioate. This provides a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. While dndA is clearly transcribed independently, dndB-E constitute an operon, as revealed by RT-PCR analysis. An efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these dnd genes. The Dnd- phenotype caused by specific in-frame deletion of the dndA, C, D, and E genes or the enhanced Dnd phenotype resulting from in-frame deletion of dndB could be restored by expression vectors carrying the corresponding dnd genes. Interestingly, overdosage of DndC or DndD, but not other Dnd proteins, in vivo was found to be detrimental to cell viability.ConclusionDNA phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. Overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host.
Highlights
A novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats
Identification of a minimal dnd region In an effort to precisely localize the region responsible for the Dnd phenotype and obtain unambiguous evidence on the genes involved in DNA phosphorothioation, we made a series of pHZ1900 derivatives by removing end segments from a ca. 10-kb fragment of DNA carrying some likely cis-acting elements using convenient restriction sites, identifying a core region carrying only five dnd genes
Mediated by the attP site of Streptomyces phage ØC31 present on pSET152, these vectors can site- integrate into the attB site in the chromosome of S. lividans ZX1 [9] after transfer by conjugation from E. coli ET12567/pUZ8002 into ZX1
Summary
A novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system. In contrast to other types of DNA modification, S, lividans has a site-specific and stereo-selective sulfur modification on the DNA backbone termed phosphorothioation [5,6,7]. This sulfur modification occurs between two guanine nucleotides in S.lividans [6,8]. The Dnd phenotype can be overcome by replacing Tris with Hepes in the electrophoresis buffer or by adding a certain concentration of thiourea to Tris-containing buffers [14,15]
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